The proposed research will deal with the relationship of the structure of thiamin pyrophosphate to the nature of its interaction with apoenzymes, divalent cations and substrates during the formation of holoenzymes and catalytic process. Transketolase and pyruvate decarboxylase will be investigated to determine whether TPP binding is similar in these enzymes. The specific areas to be studied include: 1. EPR, PRR and fluorescence techniques will be used to determine the strength and the order of binding of coenzymes and substrates to apotransketolase in an attempt to explain the "quasi-irreversibility" of formation of holo-transketolase. 2. The spatial relationship of coenzymes and substrates in the active site transketolase will be investigated by EPR and NMR (PRR) techniques. The paramagnetic probes for measurement of distances will include metal ion(s) and spin-label analogues of the coenzyme and the substrates. 3. The mechanism of catalysis by TPP in the pyruvate decarboxylase reaction will be studied with transition state analogues, and a search will be made for suicide inhibitors. 4. The freedom of rotation of the three-carbon intermediate in the transaldolase-catalyzed reaction will be investigated with 13C-labeled substrates. 5. Analogues of thiamin have been synthesized for study of the mechanism of catalysis by thiamin. Thiamin deficiency occurs in chronic alcoholism and may be caused by excessive amounts of tannins in marginal diets; disroders of thiamin function are associated with neurological diseases especially Wernicke's encephalopathy which is a late manifestation in chronic alcoholism, and Leigh's syndrome, a fatal neurological disease of infants and young children. Better understanding of the mechanism of thiamin interactions and function may aid in the understanding of these diseases.